Halogen-π Interactions in the Cytochrome P450 Active Site: Structural Insights into Human CYP2B6 Substrate Selectivity

Abstract

Numerous cytochrome P450 (CYP) 2B6 substrates including drugs and environmental chemicals are halogenated. To assess the role of halogen-π bonds in substrate selectivity and orientation in the active site, structures of four CYP2B6 monoterpenoid complexes were solved by X-ray crystallography. Bornyl bromide exhibited dual orientations in the active site with the predominant orientation revealing a bromine-π bond with the Phe108 side chain. Bornane demonstrated two orientations with equal occupancy; in both, the C2 atom that bears the bromine in bornyl bromide was displaced by more than 2.5 Å compared with the latter complex. The bromine in myrtenyl bromide π-bonded with Phe297 in CYP2B6, whereas the two major orientations in the active site mutant I114V exhibited bromine-π interactions with two additional residues, Phe108 and Phe115. Analysis of existing structures suggests that halogen-π interactions may be unique to the CYP2B enzymes within CYP family 2 but are also important for CYP3A enzymes.

Figure 1

Halogen-π interactions in CYP2B structures. Ligands are shown in yellow, and heme is depicted in red sticks: A, the Cl of the chlorophenyl moiety of ticlopidine coordinating Cl-π bond with the aromatic ring of F297 shown in stick representation in the rabbit CYP2B4 structure (green). B and C, demonstration of Cl-π interaction between the second amlodipine and the aromatic ring of F365(sticks) in the CYP2B4 complex (green). C, the altered orientation of amlodipine and substitution of methionine at position 365 in human CYP2B6 structure (pink) is shown in sticks.

Figure 2

Representative geometric models of halogen-π interactions: A, the C-X--π systems considered in this study extends from bromine (X) to the centroid of the aromatic residue side chain (Phe, Tyr, Trp and His) in the protein, with θ approximately 120° or more and α less than 60°. B, the edge-on geometry where the halogen atom (X=bromine in this study) approach the periphery of the ring with a distance range of from 3.7 Å to around 4.3 Å. The geometry is further defined by the difference between the halogen atom and the centroid or the nearest atom of the aromatic ring (r-r'>0.3 Å).

Figure 3

Structures of CYP2B6-bornyl bromide complex (green) and CYP2B6-bornane complex (pink). Heme is shown as red sticks. Bornyl bromide and bornane are shown as blue sticks. A, an unbiased F_o-F_c omit map in orange mesh at 3σ contour level calculated prior to inclusion of ligand demonstrates the well-defined electron density of bornyl bromide that corresponds to one large lobe with a bromine up orientation, and another minor lobe with bromine down orientation located near the heme iron. B, the bromine of bornyl bromide forming π bonds with the aromatic side chain of F108 is shown at appropriate distances and angles. C, two orientations were modeled with bornane in equal occupancy that were distinct from bornyl bromide. D, the location of C2 atom that bears the bromine atom in the major conformer of bornyl bromide is shown in the respective conformer of bornane. For clarity, the representative sketches of bornyl bromide and bornane have been incorporated in the bottom-right corner of panels A and C.

Figure 4

Structures of CYP2B6 and CYP2B6 I114V in complex with (-)-myrtenyl bromide. A, an unbiased F_o-F_c omit map (orange mesh) at 3σ before modeling (-)-myrtenyl bromide in the CYP2B6 active site (cyan) in two orientations. The bromine up and bromine down orientations were modeled with equal occupancies in the active site. B, the bromine up orientation of (-)-myrtenyl bromide facilitates the π-bond with F297 side chain in the active site. C, the figure illustrates the two major electron density lobes in orange mesh with bromine up orientations and a minor bromine down orientation facing the heme iron in the CYP2B6 I114V structure (yellow). Compared with the CYP2B6 (-)-myrtenyl bromide complex, an additional bromine up orientation was evident in the vicinity of I114V mutation in the active site. D, the two predominant orientations of (-)-myrtenyl bromide coordinating π interactions with three crucial aromatic side chains of F108, F115 and F297 in the active site. The new orientation allows additional π –bond formation compared to the wild-type enzyme. Heme is shown in red and (-)-myrtenyl bromide in light blue sticks. The (-)-myrtenyl bromide sketch is provided in the bottom-right corner of panels A and C.